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EoE Sub Articles

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.
1. A. fumigatus&#160;Culture and Conditions
<ol>
	<li>Prepare glucose minimal agar medium as described.
	<ol>
		<li>Adjust media to pH 6.5 using 1 M sodium hydroxide (NaOH) and autoclave at 120 &#176;C for 20 min.</li>
		<li>Allow to cool to 65 &#176;C.</li>
		<li>Working in a sterile flow hood, pour 30 mL of cooled media into a T75 flask and allow it to cool with the neck of the flask resting on a 25 mL pipette to create an agar slope.</li>
	</ol>
	</li>
	<li>Streak out the dsRed Af293-Aspergillus fumigatus&#160;strain on the agar and incubate for 7 days at 37 &#176;C + 5% carbon dioxide (CO2). Two flasks will yield approximately 109&#160;conidia in 5 mL phosphate-buffered saline (PBS) pre-biotinylation.</li>
</ol>
2. A. fumigatus&#160;Labeling and Preparation
NOTE: When performing this assay for the first time, prepare the parental Af293-A. fumigatus&#160;strain. Take samples of both strains in microcentrifuge tubes and label only one of each strain as described below. This will allow one to have control conidia with no color and conidia with a single color, either dsRed or Alexa Fluor 633 (AF633), to test the setup and equipment.
<ol>
	<li>Harvest&#160;A. fumigatus&#160;conidia by immersing the culture with 30 mL PBS + 0.05% Tween-80. Filter the resulting suspension through a 40 &#181;m cell strainer into a 50 mL tube to remove hyphal fragments.</li>
	<li>Centrifuge at 805 x g for 10 min, remove the supernatant, and wash once in 20 mL of sterile PBS. Pool conidia from two plates of the same strain during the wash step.</li>
	<li>Following the washing step, resuspend the pellet in 5 mL of PBS and transfer 1 mL aliquots of conidial suspension into microcentrifuge tubes. 1 mL of suspension of each strain is usually sufficient for live cell imaging. Wrap the remaining aliquots in foil and store at 4 &#176;C.</li>
	<li>Centrifuge aliquots of conidial suspension at 9,300 x g for 10 min at room temperature and carefully remove supernatant. Resuspend the conidial pellet in 1 mL 0.05 M sodium bicarbonate (NaHCO3)&#160;pH 8.3.</li>
	<li>Prepare 10 mg biotin/200 &#181;L dimethyl sulfoxide (DMSO) stock solution by reconstituting 25 mg biotin in 500 &#181;L DMSO. Add 10 &#181;L biotin/DMSO stock solution to the microcentrifuge tube, cover it in aluminum foil, and incubate for 2 h on a rocker at 4 &#176;C.
	<ol>
		<li>Aliquot the remainder of the biotin/DMSO stock solution and freeze at -20 &#176;C for use in subsequent experiments.</li>
	</ol>
	</li>
	<li>Centrifuge for 10 min at 9,300 x g and carefully remove the supernatant. Resuspend the conidial pellet in 1 mL of 100 mM Tris (hydroxymethyl) aminomethane-hydrochloride (Tris-HCl) pH 8.0 for 1 h to deactivate free-floating biotin.</li>
	<li>Centrifuge for 10 min at 9,300 x g and carefully remove the supernatant. Wash the pellet twice with 1 mL of sterile PBS and resuspend the pellet in 1 mL of PBS.</li>
	<li>Dissolve 1 mg of Streptavidin-AF633 in 0.5 mL of PBS to make a 2 mg/mL stock solution. Add 10 &#181;L of 2 mg/mL streptavidin-AF633 per 1 mL of conidial suspension and incubate for 40 min at room temperature on a rocker covered in aluminum foil. The remaining Streptavidin-AF633 can be frozen in aliquots for use in subsequent experiments.</li>
	<li>Centrifuge for 10 min at 9,300 x g and carefully remove supernatant. Resuspend the conidial pellet in 1 mL of PBS and count the conidia using a hemocytometer at a 1:1,000 dilution (1:100 and then 1:10). Adjust the conidial concentration to 3.6 x 106/mL with CO2-independent media, wrap in foil and store at 4 &#176;C. 
	NOTE: The conidia are now labeled with AF633 and will be referred to as FLuorescent&#160;Aspergillus&#160;REporter (FLARE) conidia.</li>
	<li>Optional:&#160;If stimulation with swollen conidia is required, after counting conidia (step 2.9), dilute conidia to 7.2 x 106/mL in yeast nitrogen base (YNB) medium and add 500 &#181;L conidial suspension to 4.5 mL YNB medium in an autoclaved 50 mL Erlenmeyer flask. Cover and place the flask in a shaking incubator (200 rpm at 37 &#176;C) for 6 h.
	<ol>
		<li>Transfer the medium to a 15 mL tube. Centrifuge at 805 x g for 10 min at room temperature and wash once in PBS. Centrifuge again and resuspend pellet in 1 mL CO2&#160;independent medium, giving 3.6 x 106&#160;conidia/mL. 
		NOTE:&#160;Conidia frequently clumps after they become swollen making accurate counting difficult, it is advised to calculate with the counted numbers of resting conidia used.</li>
	</ol>
	</li>
</ol>
3. Isolation of Human Neutrophils and Monocytes
<ol>
	<li>Draw 20 mL of venous blood from healthy volunteers into two 10 mL blood tubes containing ethylenediaminetetraacetic acid (EDTA). For each donor separately, pour 20 mL blood into a 50 mL tube and dilute with 15 mL PBS.</li>
	<li>Underlay the blood with 15 mL of a lymphocyte isolation solution (density 1.077 g/mL) with a syringe and iron needle. Place the needle at the bottom of the tube and gently force the lymphocyte isolation solution out. Centrifuge at 630 x g for 20 min with no brake and low acceleration.</li>
	<li>Prepare PBS, chilled on ice.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: Steps 3.4 and 3.5 must be followed simultaneously to generate monocytes (3.4) and neutrophils (3.5).</li>
	<li>Using a pastette, harvest the peripheral blood mononuclear cells (PBMC) layer. This is the layer in the center between the yellow serum (upper) and the transparent lymphocyte isolation solution (lower) layer, and transfer into a fresh 50 mL tube.
	<ol>
		<li>Fill the tube with the PBMC up to 50 mL with cold PBS and centrifuge at 582 x g for 10 min. Remove the supernatant and wash twice with cold PBS and resuspend in 1 mL of PBS per 20 mL of donor blood used initially.</li>
		<li>Make 1:10 dilutions for counting by adding 20 &#181;L of the cell suspension to 160 &#181;L of PBS and 20 &#181;L of trypan blue. Count cells with a hemocytometer.</li>
		<li>Prepare a buffer solution by adding 26 mL of heat-inactivated fetal calf serum (FCS) and 2.1 mL of 0.5 M EDTA to 500 mL of Hank&#39;s balanced salt solution (HBSS). Centrifuge the cells for 10 min at 515 x g and resuspend in 40 &#181;L of buffer solution per 1 x 107&#160;cells.</li>
		<li>Transfer the cell suspension to a 15 mL tube and add 10 &#181;L of CD14 microbeads per 1 x 107&#160;cells, mix well, and incubate for 15 min at 4 &#176;C, making sure to mix the tubes every 5 min.</li>
		<li>Wash cells by adding 1 mL of buffer solution per 1 x 107&#160;cells and centrifuge at 515 x g for 10 min.</li>
		<li>Aspirate supernatant completely and resuspend up to 1 x 108&#160;cells in 500 &#181;L of buffer solution.</li>
		<li>Place 1 column per sample on a magnetic separator according to the manufacturer&#39;s instructions. First, wash the column once with 500 &#181;L of buffer solution.</li>
		<li>Pipette the 500 &#181;L of cell suspension through the column, wait for the column to dry, and then wash by repeating this three times with buffer solution.</li>
		<li>Place a new 15 mL tube underneath the column and take the column off the magnetic separator. Then flush the cells out of the column into the tube with 1 mL of buffer solution.</li>
		<li>Add 4 mL of buffer solution and centrifuge at 515 x g for 10 min, and then resuspend in 1 mL of Roswell Park Memorial Institute (RPMI) medium.</li>
		<li>Make 1:10 dilutions for counting by adding 20 &#181;L of the cell suspension to 160 &#181;L of PBS and 20 &#181;L of trypan blue. Count the cells with a hemocytometer.</li>
		<li>Adjust the cell concentration to 6 x 105/mL with RPMI and seed 200 &#181;L on a 35 mm glass-based imaging dish. Incubate overnight at 37 &#176;C with 5% CO2.</li>
		<li>The next day, prior to imaging, remove the RPMI and add 200 &#181;L of CO2-independent medium. 
		NOTE: These monocytes can also be differentiated into various macrophage subsets prior to imaging. If this is a technique to be explored, an excellent starting point is a recent paper by Ohradanova-Repic&#160;et al.</li>
	</ol>
	</li>
	<li>After harvesting the PBMC layer, remove all of the serum and most of the lymphocyte isolation solution, but be careful not to remove the small white band on top of the red pellet as this will contain most of the neutrophils.
	<ol>
		<li>Prepare a 10x hypotonic lysis buffer stock by adding 83 g of ammonium chloride (NH4Cl) and 10 g of potassium bicarbonate (KHCO3)&#160;in 1 L of sterile water. Store at 4 &#176;C.</li>
		<li>Dilute this stock 10x with sterile water and add it to the tubes. Then, carefully invert 3 times and incubate in ice for 15 min.</li>
		<li>Centrifuge at 394 x g for 10 min and resuspend in hypotonic lysis buffer for 10 min in ice.</li>
		<li>Centrifuge at 394 x g and wash twice with cold PBS. Resuspend in 1 mL of CO2-independent medium per donor.</li>
		<li>Make 1:10 dilutions for counting by adding 20 &#181;L of the cell suspension to 160 &#181;L of PBS and 20 &#181;L of trypan blue. Count the cells with a hemocytometer.</li>
		<li>For microscopy, add 200 &#181;L of the same cell suspension to a 35 mm glass-based imaging dish.&#160;&#160;&#160; 
		NOTE:&#160;Neutrophil imaging or flow cytometry should be initiated immediately due to their propensity to undergo apoptosis if left unstimulated. If this is not possible neutrophils should be kept on ice and used as soon as possible (within the same day).</li>
	</ol>
	</li>
</ol>
4. Live Cell Video Microscopy
NOTE: The choice of the microscope will depend upon what is available locally, but the microscope setup will need to include an inverted stage, an environmental chamber heated to 37 &#176;C and appropriate excitation/emission filters for dsRed (532/561 nm) and AF633 (633 nm).&#160;FLARE conidia do not work with the 488 nm laser in lieu of the 532/561 nm laser for dsRed. When performing this experiment for the first time, use the control conidia with no color and single color to test for background fluorescence and bleed-through between the dsRed and AF633 channels.
<ol>
	<li>Turn on the microscope heater prior to the experiment and allow sufficient time for the environmental control chamber to warm to 37 &#176;C. The time taken for chamber temperature to stabilize will vary for different microscope setups.</li>
	<li>Turn on the microscope and computer, and load the imaging software. Mount the imaging dish on the microscope stage and adjust the focus to find the human neutrophils or monocytes.</li>
	<li>For dsRed and AF633, set the laser power to 10% and exposure time to 1 s in the acquisition settings.</li>
	<li>Remove the imaging slide and add 100 &#181;L of 3 x 106/mL FLARE conidial suspension to the appropriate wells (total volume 300 &#181;L/well). Record the time that FLARE conidia are added to the dish.</li>
	<li>Return the slide to the stage and adjust camera sensitivity for dsRed and AF633 to clearly see the conidia but not overexpose the image. Set up a stage points list if multi-point acquisition is required.</li>
	<li>Commence imaging when all points are in focus, the channels are optimized and the cycle time and duration is set. Cycle time and duration of imaging depend on the experimental question. The goal is to keep the cycle time as short as possible (&#8804;2 min) with 1 min allowing for in-depth analysis of uptake dynamics.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Glycerol</td>
			<td>Sigma</td>
			<td>G6279</td>
			<td>http://www.sigmaaldrich.com</td>
		</tr>
		<tr>
			<td>T75 flask</td>
			<td>Greiner Bio-One</td>
			<td>658 175</td>
			<td>http://www.greinerbioone.com/</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>20012-019</td>
			<td>https://www.thermofisher.com/</td>
		</tr>
		<tr>
			<td>Tween-80</td>
			<td>Fisher Scientific</td>
			<td>10592955</td>
			<td>https://nl.fishersci.com/</td>
		</tr>
		<tr>
			<td>40 &#181;m filter</td>
			<td>Thermo Fisher Scientific</td>
			<td>22363547</td>
			<td>https://www.thermofisher.com/</td>
		</tr>
		<tr>
			<td>15 ml Falcon tube</td>
			<td>Greiner Bio-One</td>
			<td>188 261</td>
			<td>http://www.greinerbioone.com/</td>
		</tr>
		<tr>
			<td>50 ml Falcon tube</td>
			<td>Greiner Bio-One</td>
			<td>227 261</td>
			<td>http://www.greinerbioone.com/</td>
		</tr>
		<tr>
			<td>MilliQ</td>
			<td>Millipore</td>
			<td>QGARD00R1</td>
			<td>Nanopure water works similarly. http://www.merckmillipore.com/</td>
		</tr>
		<tr>
			<td>Biotin</td>
			<td>Molecular Probes</td>
			<td>B-6352</td>
			<td>https://www.thermofisher.com/uk/en/home/brands/molecular-probes.html</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D5879</td>
			<td>http://www.sigmaaldrich.com</td>
		</tr>
		<tr>
			<td>Streptavidin-AF633</td>
			<td>Molecular Probes</td>
			<td>S-21375</td>
			<td>AF633 is the only tested fluorophore so far that remains visible in phagosomes. https://www.thermofisher.com/uk/en/home/brands/molecular-probes.html</td>
		</tr>
		<tr>
			<td>EDTA blood tubes</td>
			<td>Greiner Bio-One</td>
			<td>455036</td>
			<td>Any blood tubes with an anti-coagulating agent should work. http://www.greinerbioone.com/en/start/</td>
		</tr>
		<tr>
			<td>Ficoll-Paque Plus</td>
			<td>GE Healthcare</td>
			<td>17-1440-03</td>
			<td>http://www3.gehealthcare.com/</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Thermo Fisher Scientific</td>
			<td>SV3008401</td>
			<td>https://www.thermofisher.com/</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Gibco</td>
			<td>14170112</td>
			<td>https://www.thermofisher.com/uk/en/home/brands/gibco.html</td>
		</tr>
		<tr>
			<td>CD14 microbeads</td>
			<td>Myltenyi Biotec</td>
			<td>130-050-201</td>
			<td>Other monocyte isolation methods can be used as well, we prefer this method as it gives a consistent high purity of monocytes without being labor intensive. http://www.miltenyibiotec.com/en/</td>
		</tr>
		<tr>
			<td>MS Columns</td>
			<td>Myltenyi Biotec</td>
			<td>130-042-201</td>
			<td>See comment at CD14 microbeads. http://www.miltenyibiotec.com/en/</td>
		</tr>
		<tr>
			<td>RPMI + Glutamax</td>
			<td>Gibco</td>
			<td>72400-021</td>
			<td>https://www.thermofisher.com/uk/en/home/brands/gibco.html</td>
		</tr>
		<tr>
			<td>CO2 independent medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>18045054</td>
			<td>Necessary if there is not sufficient CO2 exchange during imaging. https://www.thermofisher.com/uk/en/home</td>
		</tr>
		<tr>
			<td>&#181;-slide 8 well glass bottom</td>
			<td>Ibidi</td>
			<td>80827</td>
			<td>This is the imaging dish that we use, however any glass imaging dish of proper size should work. http://ibidi.com/</td>
		</tr>
		<tr>
			<td>UltraVIEW VoX 3D Live Cell Imaging System</td>
			<td>Perkin Elmer</td>
			<td>L7267000</td>
			<td>Includes volocity software for acquisition and analysis. http://www.perkinelmer.com/</td>
		</tr>
	</tbody>
</table>

real-time assessment of the antifungal activity of primary human immune cells

Source: Brunel, S. F. et al., <a href="https://app.jove.com/v/55444">Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus.</a>J. Vis. Exp. (2017)
This video demonstrates live cell imaging for the analysis of antifungal activity in human neutrophils using fluorescent Aspergillus reporter conidia. Initially, both red and magenta fluorescence are detected within neutrophils, while red fluorescence decreases over time, indicating the degradation of conidia and confirming the antifungal activity of neutrophils.

Real-Time Assessment of the Antifungal Activity of Primary Human Immune Cells

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...

Begin with a glass-based imaging dish containing a suspension of human neutrophils &#8212; or immune cells and visualize it under a fluorescence imaging system.
Introduce genetically modified fluorescent Aspergillus reporter conidia &#8212; or fungal spores, and capture images at regular intervals up to 6 hours.
The fungal spores express red fluorescent proteins and are externally labeled with non-degradable magenta fluorophores &#8212; helping to distinguish dead cells from live ones.
During incubation, neutrophils interact with pathogen-associated molecules on conidia through pattern recognition receptors, facilitating their engulfment within a phagosome.
Later, the phagosome fuses with the enzyme-containing lysosome, forming a phagolysosome.
Inside the phagolysosome, the reactive oxygen species and enzymes degrade conidia.
During live cell imaging, at the initial time point, intense red and magenta fluorescence within the phagosome of neutrophils indicates the presence of live conidia.
Over time, a decrease in red fluorescence with the retention of magenta fluorescence indicates the degradation of conidia, confirming the antifungal activity of neutrophils.

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57 Views. Source: Brunel, S. F. et al., Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus.J. Vis. Exp. (2017) This video demonstrates live cell imaging for the analysis of antifungal activity in human neutrophils using fluorescent Aspergillus reporter conidia. Initially, both red and magenta fluorescence are detected within neutrophils, while red fluorescence decreases over time, indicating the degradation of conidia and confirming the antifungal a...

Video: Real-Time Assessment of the Antifungal Activity of Primary Human Immune Cells - Concept

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to leukocytes.

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...

JoVE Journal

Immunology and Infection

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

Escherichia coli (E. coli) is the most common Gram-negative bacteria causing neonatal meningitis. The occurrence of bacteremia and bacterial penetration through the blood-brain barrier are indispensable steps for the development of E. coli meningitis. Reactive oxygen species (ROS) represent the major bactericidal mechanisms of neutrophils to destroy the invaded pathogens. In this protocol, the time-dependent intracellular ROS production in neutrophils infected with meningitic E. coli was quantified using fluorescent ROS probes detected by a real-time fluorescence microplate reader. This method may also be applied to the assessment of ROS production in mammalian cells during pathogen-host interactions.

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

Escherichia coli is the leading cause of neonatal Gram-negative bacterial meningitis. During the bacterial infection, reactive oxygen species produced by neutrophils play a major bactericidal role. Here we introduce a method to detect the reactive oxygen species in neutrophils in response to meningitis E. coli.

Escherichia coli (E. coli) is the most common Gram-negative bacteria causing neonatal meningitis. The occurrence of bacteremia and bacterial penetration ...

Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms

Neutrophils are the first line of defense deployed by the immune system during microbial infection. In vivo, neutrophils are recruited to the site of infection where they use processes such as phagocytosis, production of reactive oxygen and nitrogen species (ROS, RNS, respectively), NETosis (neutrophil extracellular trap), and degranulation to kill microbes and resolve the infection. Interactions between neutrophils and planktonic microbes have been extensively studied. There have been emerging interests in studying infections caused by biofilms in recent years. Biofilms exhibit properties, including tolerance to killing by neutrophils, distinct from their planktonic-grown counterparts. With the successful establishment of both in vitro and in vivo biofilm models, interactions between these microbial communities with different immune cells can now be investigated. Here, techniques that use a combination of traditional biofilm models and well-established neutrophil activity assays are tailored specifically to study neutrophil and biofilm interactions. Wide-field fluorescence microscopy is used to monitor the localization of neutrophils in biofilms. These biofilms are grown in static conditions, followed by the addition of neutrophils derived from human peripheral blood. The samples are stained with appropriate dyes prior to visualization under the microscope. Additionally, the production of ROS, which is one of the many neutrophil responses against pathogens, is quantified in the presence of a biofilm. The addition of immune cells to this established system will expand the understanding of host-pathogen interactions while ensuring the use of standardized and optimized conditions to measure these processes accurately.

Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms

The present protocol describes the study of neutrophil-biofilm interactions. Staphylococcus aureus biofilms are established in vitro and incubated with peripheral blood-derived human neutrophils. The oxidative burst response from neutrophils is quantified, and the neutrophil localization within the biofilm is determined by microscopy.

Neutrophils are the first line of defense deployed by the immune system during microbial infection. In vivo, neutrophils are recruited to the site of ...

Real-Time Assessment of the Antifungal Activity of Primary Human Immune Cells

Transcript

Real-Time Assessment of the Antifungal Activity of Primary Human Immune Cells

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms